PROJECT SUMMARY Alterations in chromatin regulators that target histone post-translational modifications (PTMs) are associated with diverse human pathologies, most notably cancer. The ability to quantitatively assess disease- associated histone PTMs and their response to therapeutic intervention could have broad clinical applications, including drug discovery, clinical trial design, and personalized treatment regimens. Chromatin Immunoprecipitation (ChIP), the current leading approach to map epigenetic marks in vivo, yields only semi- quantitative data, limiting its clinical use. EpiCypher has recently developed the first quantitative ChIP platform, termed CAP-ChIP (Calibration and Antibody Profiling), which uses PTM-defined recombinant designer nucleosomes (dNucs) at a range of concentrations as DNA barcoded ?spike-in? calibration standards. However, the potential application of CAP-ChIP in pre-clinical / clinical settings remains unexplored. Here, EpiCypher is applying CAP-ChIP to quantitatively monitor histone PTM changes in response to epigenetic-targeted therapy in tumor and healthy peripheral blood mononuclear cells (PBMCs) in a preclinical model. The innovation of this project stems from the application of defined calibration standards to enable quantitative ChIP-seq with sufficient sensitivity, precision, and specificity to resolve biologically-relevant changes induced by epigenetic inhibitor treatment. The development of quantitative response measurements for epigenetically-targeted therapies could accelerate their pre- and post- clinical evaluation. For these studies we will focus on histone methyltransferase EZH2 (the catalytic subunit of the PRC2 complex) and its enzymatic product, H3K27me3. EZH2 mutation and dysregulation have been linked to a wide array of cancers, and its inhibition slows cancer cell proliferation in vitro and in vivo. As such, EZH2 has been a major focus of therapeutic development, with several inhibitors already in Phase I/II clinical trials, making it an excellent target for this proof of concept study. We will specifically focus on diffuse large B cell lymphoma (DLBCL), which is responsive to EZH2 inhibition, and a disease in which EZH2 inhibitors (e.g. Tazemetostat) are in clinical trials. CAP-ChIP-Seq will first be applied to explore the H3K27me3 landscape in cultured DLBCL cells -/+ treatment with an EZH2- specific inhibitor (Tazemetostat from Epizyme; Aim 1). We will then quantitatively evaluate the H3K27me3 landscape in xenograft DLBCL tumors from mice treated with EZH2 inhibitor (Aim 2). Since many cancers cannot be easily biopsied to assess treatment response, we will also test the application of CAP-ChIP to PBMCs to determine to how PBMC chromatin changes correlate with those in the lymphoma. In Phase II, we will scale up manufacturing and commercialize CAP-ChIP kits to selected high value drug targets for preclinical drug development: e.g. LSD1 (H3K4me2/3 and H3K9me2/3), and SETD8 (H4K20me1). In addition, EpiCypher will partner with pharmaceutical companies with active EZH2 drug development programs to develop pharmacodynamic assays and possible response-based biomarkers.